FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada ...€¦ · Table 3. Performance...
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Efficacy of Recovery and Detection of Sublethally Stressed Listeria Using Different Enrichment Media: from in vitro to in situ Sergiy Olishevskyy 1 , Carolina Mejia-Wagner 1 , Ariane Favreau 1 , Alex Eyraud 1 and Michael Giuffre 2 1 FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada 2 FoodChek Systems Inc., Calgary, Alberta, Canada The ability of Listeria monocytogenes to proliferate in various foods at refrigeration temperatures and survive even after deep freezing makes the occurrence of this foodborne pathogen in ready-to-eat (RTE) foods of particular concern. It is especially threatening to the deli meat and dairy industries if fast and reliable detection methods are not applied. Since L. monocytogenes in RTE food can be present at low concentration with sub-lethal injury during food processing, an enrichment step is crucial to resuscitate injured cells and allow sufficient growth for detection. The goal of the study was to determine the effectiveness of selective enrichment broth for the recovery and detection of sublethally stressed L. monocytogenes in ready-to-eat foods. RT-PCR Detection Culture Detection Time (Hours) OD 600nm 0 2 4 6 8 10 12 14 16 18 20 22 24 0.0 0.2 0.4 0.6 Time (Hours) OD 600nm 0 2 4 6 8 10 12 14 16 18 20 22 24 0.0 0.2 0.4 0.6 Fig. 1. Growth of uninjured (A) and sublethally heat-injured (B) L. monocytogenes A. B. Table 1. Growth Kinetics Parameters of L. monocytogenes Bacterium Kinetics Parameter Actero Listeria BLEB UVM Uninjured Listeria LPD 9.3 ± 0.41 12.2 ± 0.56 ** 16.4 ± 0.75 *** GR 0.352 ± 0.056 0.274 ± 0.020 * ND Heat-injured Listeria LPD 11.1 ± 0.94 16.0 ± 1.26 ** 18.1 ± 0.88 ** GR 0.368 ± 0.061 ND ND Table 2. Food Matrices and Inoculating Microorganisms Food Matrix Sample Size L. monocytogenes Background Flora, log 10 CFU/g Serotype Origin Stress Pasteurized Milk 25 g 4c Beef manure Heat < 2.0 Soft Cheese 25 g 1/2a Sheep’s head None 7.5–8.0 Frankfurter 125 g 1/2a Milk Heat < 2.0 Smoked Turkey 125 g 3b Raw turkey Heat 4.7–5.0 Cured Ham 125 g 1/2a Animal tissues Heat < 2.0 Table 3. Performance Parameters for Detection of L. monocytogenes Using BAX System RT-PCR Assay in Dairy and Deli Meat Products Enriched with Actero Listeria Food Matrix Total Tested P N FP FN Relative Sensitivity, % Relative Specificity, % FP Rate, % FN Rate, % Test Efficacy, % Pasteurized milk 60 43 16 0 1 97.7 100 0.0 2.3 98.3 Soft cheese 60 36 24 0 0 100 100 0.0 0.0 100 Frankfurter 30 19 11 0 0 100 100 0.0 0.0 100 Smoked turkey 30 12 18 0 0 100 100 0.0 0.0 100 Cure ham 30 16 14 0 0 100 100 0.0 0.0 100 TOTAL: 210 126 83 0 1 99.2 100 0.0 0.8 99.5 Notes: P – positive, N – negative, FP – false positive, FN – false negative. Fig. 3. Recovery of L. monocytogenes Using Actero Listeria Notes: BLEB Actero Listeria UVM Culture Conditions • Initial Inoculum – 20 CFU/well • Medium Volume – 200 μL • Temperature – 35°C • Time – 22 hours Fig 2. Method Comparison Matrix Studies Flowchart Food Samples Dairy Products Deli Meat Products Actero Listeria Method US FDA BAM10 Method 125 g sample 25 g sample 25 g sample 500 - 1125 mL Actero Listeria 24 - 26hrs, 35 ° C 225 mL BLEB 48hrs, 30 ° C 150mL Actero Listeria 22hrs, 35 ° C Morphological, biochemical and serological identification up to 72 hrs BAX System Assay, Listeria Genus 2 hrs BAX System Assay, L. monocytogenes 2 hrs Total Time 24-28 hrs Total Time up to 120 hrs Direct Plating Rapid’L.mono / MOX 24 hrs Total Time 46-50 hrs Total Time up to 120 hrs USDA FSIS MLG 8.10 Method 125 g sample 1125 mL , UVM 23 - 26hrs, 30 ° C 10 mL , MOPS - BLEB 18 - 26hrs, 35 ° C Morphological, biochemical and serological identification up to 72 hrs Notes: LPD – Lag Phase Duration; GR – Growth Rate. ND – Not Determined: Kinetics parameters could not be defined because all growth stages were not observed during the incubation period. * P<0.05, ** P<0.01, *** P<0.001 as compared to the corresponding values obtained for Actero Listeria. The performance parameters of relative sensitivity, relative specificity, false positive rate (FP), false negative rate (FN), and test efficacy were calculated for the Actero Listeria method and are presented in Table 3. Only one false negative outcome out of 210 analyzed samples was observed. Food Samples 98 55 *** 42 85 Actero Negative Outcomes Actero Positive Outcomes RM Positive Outcomes RM Negative Outcomes Actero Listeria shows shorter lag phase and higher growth rate for L. monocytogenes as compared to the reference method media. Enrichment with Actero Listeria results in a rapid and efficient repair of growing capacities of sublethally-injured L. monocytogenes. Deli meat and dairy samples were artificially contaminated with healthy or sub-lethally heat-stressed L. monocytogenes belonging to different serotypes (Table 2) and stabilized for 48– 72 hours at 2–8°C. Samples were enriched in Actero™ Listeria Enrichment Media, then processed using Hygiena’s BAX® System Real-Time PCR Assays for L. monocytogenes and Listeria Genus as well as by direct plating. A total of 420 artificially contaminated food samples were examined to evaluate performance of the candidate method in comparison with the USDA-FSIS or US FDA reference methods (Fig. 2). Overall, 140 fractionally contaminated food samples containing low numbers of sublethally injured L. monocytogenes and tested by the alternative method using Actero Listeria yielded 40% more positive results as compared to the samples analysed by the USDA-FSIS or US FDA methods. Single step enrichment with Actero Listeria allows for significantly reducing incubation time due to efficacious recovery of injured L. monocytogenes. BAX System Assays show high accuracy and reliability for detection of L. monocytogenes in deli meat and dairy samples enriched with Actero Listeria. Performance of the Actero Listeria method for deli meat and dairy products is superior to the reference methods. In situ Matrix Studies Method Principle Introduction In vitro Culture Studies Conclusions *** P<0.001 as compared to the corresponding values obtained for Actero Listeria.
Transcript of FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada ...€¦ · Table 3. Performance...
Efficacy of Recovery and Detection of Sublethally Stressed
Listeria Using Different Enrichment Media: from in vitro to in situ
Sergiy Olishevskyy1, Carolina Mejia-Wagner1, Ariane Favreau1, Alex Eyraud1 and Michael Giuffre2
1FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada 2FoodChek Systems Inc., Calgary, Alberta, Canada
The ability of Listeria monocytogenes to proliferate in various foods at refrigerationtemperatures and survive even after deep freezing makes the occurrence of this foodborne
pathogen in ready-to-eat (RTE) foods of particular concern. It is especially threatening to thedeli meat and dairy industries if fast and reliable detection methods are not applied. SinceL. monocytogenes in RTE food can be present at low concentration with sub-lethal injury
during food processing, an enrichment step is crucial to resuscitate injured cells and allowsufficient growth for detection.
The goal of the study was to determine the effectiveness of selective enrichment broth for
the recovery and detection of sublethally stressed L. monocytogenes in ready-to-eat foods.
RT-PCR Detection Culture Detection
T im e (H o u rs )
OD
60
0n
m
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
0 .0
0 .2
0 .4
0 .6
T im e (H o u rs )
OD
60
0n
m
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
0 .0
0 .2
0 .4
0 .6
Fig. 1. Growth of uninjured (A) and sublethally heat-injured (B) L. monocytogenes
A. B.
Table 1. Growth Kinetics Parameters of L. monocytogenes
Bacterium Kinetics Parameter Actero Listeria BLEB UVM
Uninjured ListeriaLPD 9.3 ± 0.41 12.2 ± 0.56** 16.4 ± 0.75***
GR 0.352 ± 0.056 0.274 ± 0.020* ND
Heat-injured ListeriaLPD 11.1 ± 0.94 16.0 ± 1.26** 18.1 ± 0.88**
GR 0.368 ± 0.061 ND ND
Table 2. Food Matrices and Inoculating Microorganisms
Food MatrixSample
Size
L. monocytogenesBackground Flora,
log10 CFU/gSerotype Origin Stress
Pasteurized Milk 25 g 4c Beef manure Heat < 2.0
Soft Cheese 25 g 1/2a Sheep’s head None 7.5–8.0
Frankfurter 125 g 1/2a Milk Heat < 2.0
Smoked Turkey 125 g 3b Raw turkey Heat 4.7–5.0
Cured Ham 125 g 1/2a Animal tissues Heat < 2.0
Table 3. Performance Parameters for Detection of L. monocytogenes Using BAX SystemRT-PCR Assay in Dairy and Deli Meat Products Enriched with Actero Listeria
Food MatrixTotal
TestedP N FP FN
Relative Sensitivity,
%
Relative Specificity,
%
FP Rate, %
FN Rate, %
Test Efficacy, %
Pasteurized milk 60 43 16 0 1 97.7 100 0.0 2.3 98.3
Soft cheese 60 36 24 0 0 100 100 0.0 0.0 100
Frankfurter 30 19 11 0 0 100 100 0.0 0.0 100
Smoked turkey 30 12 18 0 0 100 100 0.0 0.0 100
Cure ham 30 16 14 0 0 100 100 0.0 0.0 100
TOTAL: 210 126 83 0 1 99.2 100 0.0 0.8 99.5
Notes: P – positive, N – negative, FP – false positive, FN – false negative.
Fig. 3. Recovery of L. monocytogenes Using Actero Listeria
Notes:
T im e (H o u rs )
OD
60
0n
m
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
0 .0
0 .2
0 .4
0 .6A c te ro
T ML is te r ia
2 4 L is te r ia E n r ic h m e n t B ro th (O x o id )
U VM
L E B O xo id
B L E B (O x o id )
A N S R (N e o g e n )
BLEB
Actero Listeria
T im e (H o u rs )
OD
60
0n
m
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
0 .0
0 .2
0 .4
0 .6A c te ro
T ML is te r ia
B L E B (O x o id )
T im e (H o u rs )
OD
60
0n
m
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
0 .0
0 .2
0 .4
0 .6A c te ro
T ML is te r ia
2 4 L is te r ia E n r ic h m e n t B ro th (O x o id )
U VM
L E B O xo id
B L E B (O x o id )
A N S R (N e o g e n )
UVM
Culture Conditions
• Initial Inoculum – 20 CFU/well• Medium Volume – 200 µL• Temperature – 35°C• Time – 22 hours
Fig 2. Method Comparison Matrix Studies Flowchart
Food Samples
Dairy ProductsDeli Meat Products
Actero Listeria Method US FDA BAM10 Method
125 g sample 25 g sample25 g sample
500-1125 mL
Actero Listeria
24-26hrs, 35°C
225 mL
BLEB
48hrs, 30°C
150mL
Actero Listeria
22hrs, 35°C
Morphological, biochemical and
serological identification
up to 72 hrs
BAX System Assay,
Listeria Genus
2 hrs
BAX System Assay,
L. monocytogenes
2 hrs
Total Time
24-28 hrs
Total Time
up to 120 hrs
Direct Plating
Rapid’L.mono / MOX
24 hrs
Total Time
46-50 hrs
Total Time
up to 120 hrs
USDA FSIS MLG 8.10 Method
125 g sample
1125 mL, UVM
23-26hrs, 30°C
10 mL, MOPS-BLEB
18-26hrs, 35°C
Morphological, biochemical and
serological identification
up to 72 hrs
Notes: LPD – Lag Phase Duration; GR – Growth Rate.ND – Not Determined: Kinetics parameters could not be defined because all growth stages were not observed during the incubation period.*P<0.05, **P<0.01, ***P<0.001 as compared to the corresponding values obtained for Actero Listeria.
The performance parameters of relative sensitivity, relative specificity, false positiverate (FP), false negative rate (FN), and test efficacy were calculated for the ActeroListeria method and are presented in Table 3. Only one false negative outcome out of210 analyzed samples was observed.
Food Samples
9855
***
4285
Actero Negative Outcomes
Actero Positive Outcomes
RM Positive Outcomes
RM Negative Outcomes
A c te r o P o s it iv e O u t c o m e s
A c te r o N e g a t iv e O u tc o m e s
R M P o s it iv e O u tc o m e s
R M N e g a t iv e O u tc o m e s
Actero Listeria shows shorter lag phase and higher growth rate for L. monocytogenes as compared to the reference method media.
Enrichment with Actero Listeria results in a rapid and efficient repair of growing capacities of sublethally-injured L. monocytogenes.
Deli meat and dairy samples were artificially contaminated with healthy or sub-lethally heat-stressed L. monocytogenes belonging to different serotypes (Table 2) and stabilized for 48–72 hours at 2–8°C. Samples were enriched in Actero™ Listeria Enrichment Media, then processed using Hygiena’s BAX® System Real-Time PCR Assays for L. monocytogenes and ListeriaGenus as well as by direct plating. A total of 420 artificially contaminated food samples were examined to evaluate performance of the candidate method in comparison with theUSDA-FSIS or US FDA reference methods (Fig. 2).
Overall, 140 fractionally contaminated food samples containing low numbers ofsublethally injured L. monocytogenes and tested by the alternative method using ActeroListeria yielded 40% more positive results as compared to the samples analysed by theUSDA-FSIS or US FDA methods.
Single step enrichment with Actero Listeria allows for significantly reducingincubation time due to efficacious recovery of injured L. monocytogenes.
BAX System Assays show high accuracy and reliability for detection ofL. monocytogenes in deli meat and dairy samples enriched with Actero Listeria.
Performance of the Actero Listeria method for deli meat and dairy products issuperior to the reference methods.
In situ Matrix Studies
Method PrincipleIntroduction
In vitro Culture Studies
Conclusions
***P<0.001 as compared to the corresponding valuesobtained for Actero Listeria.
